Interneurons in the spinal cord are integral components of the neural networks that control posture and movement. We have characterized a number of embryonic interneuron (IN) cell types in the spinal cord with the long term goal of defining their functional contribution to the neural circuitry that controls locomotion. These studies have led to the identification of two classes of inhibitory spinal neurons, V1 and V2B neurons, both of which project ipsilaterally within the ventral horn. Our preliminary evidence indicates that these IN cell types connect to motor neurons and other CPG neurons. In this proposal we will use a combination of genetic, anatomical and electrophysiological approaches to characterize these two cell types and determine their functions in locomotor behaviors. We will perform whole cell recordings on V1 INs to delineate their cellular properties. We will ask whether subsets of V1 INs exhibit specificity in their synaptic connections with motor neurons. We will test our hypothesis that V1 INs provide early cycle inhibition during locomotion to motor neurons. The function of the V1 INs in awake behaving animals will be examined using a novel system that we have developed for acutely silencing neurons in mice. Our analysis of the V2B IN population will focus on characterizing the morphology, neurotransmitter phenotype and connectivity of these neurons. We will determine whether V2B INs are rhythmically active during locomotion, and are thus likely candidates for providing phasic ipsilateral inhibition to other locomotor neurons. Finally, we will test our hypothesis that V2B INs play an essential role in securing flexor-extensor motor activity in the mammalian locomotor CPG. These studies will provide valuable insights into the development and organization of locomotor circuits in the mammalian spinal cord. More importantly, they will lay the groundwork for strategies to treat spinal cord injury and degeneration disorders that affect normal voluntary movements.